H+/K+ ATPase–Dependent Changes in Retinal Pigment Epithelial (RPE) Physiology

Abstract

Abstract: : Purpose: To identify an H⁺/K⁺ ATPase (HK) in bovine and fetal human RPE and study physiological changes produced by alterations in its activity. Methods: PCR and immunofluorescence techniques were used to localize the HK in bovine and cultured human RPE preparations. A ratiometric dye (BCECF–AM) was used to measure pH_i. A capacitance probe technique was used to measure transepithelial water flow. Intracellular recordings, transepithelial potential and resistance measurements were also obtained. Results: The presence of the gastric proton pump in bovine and fetal human RPE was verified by PCR and localized to the apical plasma membrane by immunofluorescence. In bovine RPE–choroid, the TEP and R_t in control Ringer was 9.7±1.8 mV and 164.6±5.4 Ω·cm² (mean ± sem, n=5), respectively. TEP dropped to 5.9±1.2 mV and R_τ increased to 182.4±6.0 Ω·cm² after a 10–15 minute apical perfusion with Ringer containing a reversible HK antagonist (ALTANA Pharma AG), at a concentration expected to completely block HK activity. Application of the HK antagonist also decreased fluid absorption (retina–to–choroid) from 14.4±3.6 to 7.3±1.9 µl·cm^–2·hr^–1 (n=7). In primary cultures of fetal human RPE, the HK antagonist reduced fluid transport by 32±6% (n=4), and partially recovered upon washout. Application of the HK antagonist increased intracellular pH by 0.1±0.03 pH units (n=23); this effect was doubled in bovine RPE–choroid preparations bathed in zero bicarbonate Ringer. Apical Ringer with 15 mM K elevated [K]_i and this increase was blocked by the HK antagonist. Basal treatment with DIDS, but not NPPB, blocked the HK antagonist–induced changes in fluid transport, TEP and R_t(n = 2). Conclusions: These experiments suggest that the HK antagonist decreases potassium recycling at the apical membrane, chloride transport across the DIDS–sensitive basolateral chloride channels, and subsequent fluid absorption. It is assumed that the HK antagonist–induced alkalinization results from the concomitant inhibition of the apical membrane HK and another mechanism yet to be identified.

Keywords: retinal pigment epithelium • PH regulation/protons • electrophysiology: non-clinical